The long-term objectives of these proposals are to better understand the biochemical and biophysical changes that occur in the human lens with aging, and which ultimately lead to cataract. The proposed studies will focus on alpha-crystallin. In addition to being a key member of the structural proteins in the lens that are responsible for producing the needed refraction, a-crystallin is a molecular chaperone. In this capacity it helps suppress non-specific aggregation of proteins that may cause scattering and cataract, alphaBeta-crystallin, a member of the small heat-shock protein family is also involved in many age-dependent neurological/eye diseases including extracellular deposits (Drusen) that accumulate below the retinal pigment epithelium of patients with age-related macular degeneration. The proposed studies will provide new data that will be useful for achieving the ultimate goal of significantly retarding the formation of aging dependent cataract. The specific aims are: 1. Structural and functional studies. Various mutations and chemical modifications of alpha-crystallin and their effect on chaperone function will be studied with technologies such as cryo-electron microscopy and site-directed spin-labeling with electron spin resonance. 2. Studies on the effects of macromolecular crowding on protein structure and stability in the lens. These studies deal with the fact that the macromolecular concentrations in the native lens are extremely high (500 mg/ml) and may have dramatic effects on proteins structure, function, and stability. This problem has never been addressed thus far. 3. Animal models of cataract, alpha-crystallin knock-out mouse, as well as a new mouse cataract that involves a and gamma-crystallin will be studies in detail. These are excellent models for understanding the biochemical and biophysical changes that occur in age-related human cataract.